This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding caffeoyl-CoA O-methyltransferase in plants and seeds.
Plant cells and tissues can respond to mechanical, chemical or pathogen induced injury by producing various phenolic compounds including mono- or dimethoxylated lignin precursors via a complex series of biochemical reactions. These lignin precursors are eventually used by the plant to produce lignin which helps repair wounds by adding (i) hydrophobicity, a physical barrier against pathogen infection, and (ii) mechanical strength to the injured tissue (Vance, C. P. et al. (1980) Annu Rev Phytopathol 18:25-288). Biosynthesis of the mono- or dimethoxylated lignin precursors occurs, in part, by the action of two enzymes, caffeic acid 3-O-methyltransferase (COMT) and caffeoyl CoA 3-O-methyltransferase (CCOMT). Studies ave shown that the activities of these two enzymes increases prior to lignin deposition (Inoue, K. et al. (1998) Plant Physiol 117(3):761-770). Caffeoyl CoA 3-O-methyltransferase not only has been implicated in the synthesis of components during a plant""s defense response (Kxc3xchnl, T., et al. (1989) Plant Sci 60:21-25; Pakusch, A. E. et al., (1989) Arch Biochem Biophys 271:488-494) but may also play a role in cell differentiation of meristematic tissue to form tracheary elements (Ye, Z. H. et al. (1994) Plant Cell 6:1427-1439). In the final state of differentiation of tracheary elements, only the cell wall remains to form a vascular tissue through which water and solutes circulate throughout the plant. Thus lignin is a vital component of the plants cell wall architecture and plays a role in host defence and injury repair mechanisms.
Because of lignin""s importance in cell wall architecture and wound repair mechanisms there is considerable interest in the prospects for altering lignin quantity or quality by genetic engineering. For example, chemical treatments needed to remove lignin during the paper- pulping process are expensive and environmentally unfriendly. Plants with altered lignin quantity or quality could benefit this industry (Boudet, A. M. et al. (1996) Mol Breeding 2:25-39; Campbell, M. M. et al. (1996) Plant Physiol 110:3-13; Sewalt, V. J. H. et al. (1997) J Agric Food Chem 45:1977-1983; Sewalt V. J. H. et al. (1997) Plant Physiol 115:41-50). Thus, there is a great deal of interest in identifying the genes that encode proteins involved in the production of lignin in plants. Amino acid sequences of caffeoyl CoA 3-O-methyltransferase and encoding nucleotide sequences are available in the public domain (WO 9910498-A2; DE4117747-A; WO 9909188-A2; WO 9811205-A2). Caffeoyl-CoA O-methyltransferase genes appear to be part of a multigene family. These genes may be used in plant cells to control lignin production. Accordingly, the availability of nucleic acid sequences encoding all or a portion of an enzyme involved in the production of lignin would facilitate studies to better understand lignin production in plant cells and provide genetic tools to enhance or otherwise alter lignin biosynthesis which in turn could provide mechanisms to control cell wall architecture and host defence and injury repair mechanisms in plant cells.
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 50 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:8, a soybean caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:28, a soybean caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:30, a soybean caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:32, a soybean caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:34, and a wheat caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:48. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 50 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a corn caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:2. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 100 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a rice caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:16, a soybean caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:20, a wheat caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:44, and a wheat caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:46. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 100 amino acids that has at least 85% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a soybean caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:24 and a soybean caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:26. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 100 amino acids that has at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a rice caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:10, a wheat caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:36, and a wheat caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:40. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 150 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a rice caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:14. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 150 amino acids that has at least 85% identity based on the Clustal method of alignment when compared to a corn caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:6. The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 150 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a corn caffeoyl-CoA O-methyltransferase polypeptide of SEQ ID NO:42. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
It is preferred that the isolated polynucleotides of the claimed invention consist of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 5, 7, 9, 13, 15, 19, 23, 25, 27, 29, 31, 33, 35, 39, 41, 43, 45, and 47 that codes for the polypeptide selected from the group consisting of SEQ ID NOs: 1, 5, 7, 9, 13, 15, 19, 2, 6, 8, 10, 14, 16, 20, 24, 26, 28, 30, 32, 34, 36, 40, 42, 44, 46, and 48. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least one of 40 (preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 5, 7, 9, 13, 15, 19, 23, 25, 27, 29, 31, 33, 35, 39, 41, 43, 45, and 47 and the complement of such nucleotide sequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a caffeoyl-CoA O-methyltransferase polypeptide of at least 50 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:8, 28, 30, 32, 34, and 48. The present invention also relates to a caffeoyl-CoA O-methyltransferase polypeptide of at least 50 amino acids comprising at least 95% homology based on the Clustal method of alignment compared to a polypeptide f SEQ ID NO:2. The present invention also relates to a caffeoyl-CoA O-methyltransferase polypeptide of at least 100 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs: 16, 20, 44, and 46. The present invention also relates to a caffeoyl-CoA O-methyltransferase polypeptide of at least 100 amino acids comprising at least 85% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:24 and 26. The present invention also relates to a caffeoyl-CoA O-methyltransferase polypeptide of at least 100 amino acids comprising at least 90% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:10, 36, and 40. The present invention also relates to a caffeoyl-CoA O-methyltransferase polypeptide of at least 150 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide f SEQ ID NO:14. The present invention also relates to a caffeoyl-CoA O-methyltransferase polypeptide of at least 150 amino acids comprising at least 85% homology based on the Clustal method of alignment compared to a polypeptide f SEQ ID NO:6. The present invention also relates to a caffeoyl-CoA O-methyltransferase polypeptide of at least 150 amino acids comprising at least 95% homology based on the Clustal method of alignment compared to a polypeptide f SEQ ID NO:42.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a caffeoyl-CoA O-methyltransferase polypeptide in a plant cell, the method comprising the steps of:
constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention;
introducing the isolated polynucleotide or the isolated chimeric gene into a plant cell;
measuring the level of a caffeoyl-CoA O-methyltransferase polypeptide in the plant cell containing the isolated polynucleotide; and
comparing the level of a caffeoyl-CoA O-methyltransferase polypeptide in the plant cell containing the isolated polynucleotide with the level of a caffeoyl-CoA O-methyltransferase polypeptide in a plant cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a caffeoyl-CoA O-methyltransferase polypeptide gene, preferably a plant caffeoyl-CoA O-methyltransferase polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 40 (preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 5, 7, 9, 13, 15, 19, 23, 25, 27, 29, 31, 33, 35, 39, 41, 43, 45, and 47 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode all or a portion of a caffeoyl-CoA O-methyltransferase amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a caffeoyl-CoA O-methyltransferase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.